Effects of Charcoal as a Soil Conditioner on Citrus Growth and
Transcript of Effects of Charcoal as a Soil Conditioner on Citrus Growth and
J. Japan. Soc. Hort. Sci. 63(3) : 529-535. 1994.
Effects of Charcoal as a Soil Conditioner on Citrus Growth and
Vesicular-Arbuscular Mycorrhizal Development
Takaaki Ishii1 and Kazuomi Kadoya2
I Faculty of Education , Ehime University, Matsuyama, Ehime 790 2 College of Agriculture
, Ehime University, Matsuyama, Ehime 790
Summary
Effects of several kinds of charcoal applied to soil on citrus growth and vesicular-arbuscular mycorrhizal (VAM) development were investigated. Satsuma mandarin (Citrus
unshiu Marc.) trees on trifoliate orange (Poncirus trifoliata Raf.) rootstocks were trans-planted to root boxes using the soil mixed with charcoal derived from rice husk, citrus juice sediment or western spruce bark. The trees were inoculated with the spores of Glomus fasciculatum (Thaxter) Gerdemann and Trappe emend. Walker and Koske. Elongation of the roots in the charcoal treatments was more vigorous than that in the charcoal-free control. The fresh weigths of the root, shoot and the whole tree increased in response to charcoal application. The intensity of VAM infection in any charcoal treatment was higher than that in the control. In particular, the percentage of the infection in the rice husk charcoal
plot was 41.5 and P concentration in the leaf exceeded that of the control. In a Citrus iyo orchard, the percentage of VAM infection was 52% in the rice husk
charcoal plot, the highest among plots. The intensity in the Bahia grass (Paspalum notatum Flugge.) plot was next, followed by the third highest rate found in the abandoned
plot which had not been cultivated in recent years. The lowest percentage of VAM infec-tion was in a clean-culture plot. A microscopic observation also revealed that in a charcoal-treated plot there were many sites where VAM fungi infected the root.
Introduction
A classic book written in Japan (Miyazaki,
1697) explained that soybean plants vigorously
flourished with a minimum of care when their seeds were sowed with charcoal. Recently, Ogawa
(1987) reported that charcoal applied to the soil could stimulate the activity of soil microorganisms
and promote the formation of root nodules and
vesicular-arbuscular mycorrhizae in soybean roots.
VAM symbioses are exceptionally common
among terrestrial flowering plants (Harley and Harley, 1987). Among these plants, there is a wide
range of dependency on VAM fungi for plant
growth. Citrus is also infected by several kinds of VAM fungi and is considered highly dependent on
them (Dixon et al., 1988; Edriss et al., 1984; Ishii
et al., 1992b; Menge et al., 1978; Nemec, 1979). These fungi improve mineral nutrition of the host
by increasing P uptake from a P deficient soil
(Antunes and Cardoso, 1991; Ferguson and Menge, 1986; Graham and Timmer, 1985; Krikun
and Levy, 1980; Nemec, 1979; Tang et al., 1984). Higher concentration of minor elements, especially
Zn (Krikun and Levy, 1980) and Cu (Timmer and
Leyden, 1980), were also observed after an inocu-lation with VAM fungi. Because the fungi pro-
vided essential elements for citrus growth, the in-
fected trees could grow more rapidly and appeared healthier than non-infected trees. This phe-
nomenon was especially noticeable in soils of low
fertility (Nemec, 1979). Furthermore, VAM fungi inoculation may increase tolerance to water stress
by regulating stomatal opening through hormone
synthesis (Graham et al., 1987). In our reports,
the photosynthesis and transpiration rates of VAM fungi-infected satsuma mandarin trees grow-
Received for publication 7 February 1994.
Parts of this paper were presented at the 1989 Spring
Meeting and 1990 Spring Meeting of the Japanese Society
for Horticultural Science.
529
530 T. Ishii and K. Kadoya
ing in P-deficient soil surpassed those of non-VAM
trees stressed by high temperatures in August
(Shrestha et al., 1992). Interestingly, an inocula-tion of VAM fungi improved the fruit quality of
satsuma mandarin trees. In particular, it enhanced the Hunter's a/b value of peel color and the sugar
content in juice (Ishii et al., 1992b). In the citrus
orchards where high quality fruit was produced, the percentage of VAM infection in the root was
very high (Shrestha et al., 1993).
On the other hand, phytotoxic substances exist in the bark and sawdust extracts from several
woody forest species, especially in the bark ex-tracts of hinoki cypress (Chamaecyparis obtusa
Sieb. et Zucc. ex Endl.) which are condensed tan-
nins (Ishii and Kadoya, 1993). In soils to which unfermented organic matter were added, ethylene
has often been detected at concentrations high
enough to inhibit the growth of citrus trees (Ishii and Kadoya, 1984). The problem of growth inhibi-
tion is solved, however, if the organic matter with
phytotoxic substances is first transformed into charcoal and then used as a soil amendment.
The purpose of this study is to investigate
effects of several kinds of charcoal applied to soil on citrus growth and VAM development.
Materials and Methods
Experiment 1. Effects of charcoal application on citrus
growth and VAM development
In this experiment, we examined the effects of
charcoal application on the growth and VAM de-velopment of 'Aoshima' satsuma mandarin trees on
trifoliate orange rootstocks for two years. In early April of 1988, three two-year-old satsuma man-
darin trees per plot were transplanted individually to root boxes (40 cm × 40 cm × 40 cm) containing
the mixtures of river sand and a specific charcoal. Before planting, the roots were carefully washed
to remove the soil which had nourished the trees.
The control soil lacked charcoal. The charcoal used was made by using a chimney (15 cm in dia-
meter and 1.8 m in length) with some holes for
aeration or an oil drum (200 liter) equipped with
a chimney (15 cm in diameter and 1.8 m in length). The charcoal sources were rice husk, cit-
rus juice sediment and western spruce bark. The
charcoal derived from western spruce bark was
broken into 5 ~ 10 mm pieces. The sand and char-
coal were mixed in a proportion of 50 to 1 by
weight. The pH and electric conductivity (EC) of
the mixtures were measured with a pH meter and
an EC meter, respectively.
Two months after planting, the roots were
treated with 50 g fresh weight of soil inoculum con-
taining 300 ~ 340 spores of Glomus fasciculatum .
The inoculum was obtained from greenhouse pot
cultures of Bahia grass inoculated with Glomus
fasciculatum originally isolated from citrus
orchards in Matsuyama city, Ehime prefecture,
Japan. In 1988, each tree was fertilized with 6.4 g
of N, 3.2 g of P, and 3.8 g of K per annum from a
mixture of ammonium sulfate, calcium phosphate,
and potassium sulfate, respectively; the P and K
contents in the charcoal were first deducted. The
control trees were supplied with 5 g of calcium
carbonate-magnesium sulfate mixture (8 : 2 by
weight) to improve the soil pH. A Hoagland minor
element solution (1 liter/tree) was administered to
each trees. In 1989, P was excluded.
Roots appearing on the glass plates of root boxes
were traced onto transparent plastic sheets from
which the root lengths were measured, using a
personal computer equipped with an image proc-
essor (NEC mediagraph MG -10 with a stylus pen,
Tokyo, Japan) and a special software program.
In early December of 1989, the trees were re-
moved from the root boxes, and then the total,
root, and shoot fresh weights were measured. For
the determination of leaf P, leaf samples were
ashed at 550 •Ž overnight, and the residues were
dissolved in 2.4 N HC]. The P content was meas-
ured colorimetrically by the method of Deniges
(1920, 1921). Undamaged feeder rootlets were
sampled and rinsed with distilled water for a few
seconds. After the rootlets were cut into 2 - cm seg-
ments behind the growing tip, the segments were
immediately fixed in FAA (formalin: acetic acid:
50% ethanol, 13 : 5 : 200, v/v/v). Ten segments
per treatment, stained by the technique of Phillips
and Hayman (1970), were analyzed for the intensi-
ty of VAM infection by light microscopy. The per-
centage of VAM infection was calculated with the
following equation :
J. Japan. Soc. Hort. Sci. 63(3) : 529-535. 1994. 531
Experiment 2. Soil management and the intensity of VAM infection in citrus roots
In late April of 1987, 4 experimental plots of 5
trees each were prepared in a Citrus iyo (15 - year-old trees on trifoliate orange rootstock) orchard in
Matsuyama city, Ehime prefecture. The ex-
perimental plots were as follows : 1) charcoal as a soil amendment, 2) abandoned culture, 3) sod cul-
ture with Bahia grass, and 4) clean culture by us-
ing herbicides such as paraquat dichloride and N - (phosphonomethyl) glycine (3 times in a year).
The charcoal plot had two pits (60 × 60 cm in width and 40 cm in depth) circling a tree and filled
with 6 kg of rice husk charcoal. Paraquat dichlo-
ride was applied once annually. In the Bahia grass
plot, the grass was mowed once each summer. Ex-cept for the abandoned plot, the rest received 32
kg N, 23 kg P, and 25 kg K per 10 a annually. The
application of agrochemicals, such as fungicides and pesticides, followed the guidelines of disease
and pest control for Ehime prefecture. In early September of 1988, root samples were
obtained from 3 to 5 places of each plot at a depth
of 5-10 cm, and then the intensity of VAM infec-
tion was determined by the methods described above. The root structure was observed with a
scanning electron microscope (SEM, JEOL type
JSM - T200, Tokyo, Japan). The apical 20 mm of 20 elongating roots from each plot were rinsed
with distilled water for a few seconds and the
apices were immediately fixed in Karnovsky solu-tion (Karnovsky, 1965) at room temperature for
24 hr. After being dehydrated through graded
solutions of ethyl alcohol-acetone, they were divided into 4 segments in 100% acetone. These
segments were then immersed in acetone for 2 hr,
critical-point-dried, mounted on aluminum stubs with silver conducting paint, and coated with a
thin layer of gold using an ion-coater (Eiko Engineering type IB -2, Tokyo, Japan). The roots
were observed in a SEM and photographed.
Results
Experiment 1.
No differences in soil pH among treatments were
observed. The EC value of the charcoal treatments
was higher than that of the control. The EC in the
western spruce bark charcoal treatment was about
13 times higher than that of the control plot
(Table 1). This is because NaCl permeated into the bark during sea storage after being imported into
Japan from North America. Soils treated with 3 kinds of charcoal had signif-
icant effects on growth, leaf P concentration, and
VAM development in roots of satsuma mandarin
trees. About 2 months after the onset of this ex-
periment, except in the western spruce bark char-coal treatment, roots appeared on the glass plates,
and their elongation rates indicated that roots in the charcoal-treated plots were more vigorous than
ones in the control. As of November 8, 1989, the
root length in any charcoal treatment was about 1.5 times longer than that in the control. The total
fresh weights and the fresh weights of roots and
shoots increased with charcoal treatments. The
growth increments varied little among the kinds of charcoal (Table 2). The intensity of VAM infection
in any charcoal treatment was higher than that in the control; that of the rice husk charcoal treat-
ment attaining 41.5% (Table 3). Hardly any signif-icant differences in leaf P concentration among
treatments with western spruce bark charcoal, cit-
rus juice sediments charcoal and the control were
observed; but leaf P concentration in the rice husk charcoal treatment, which significantly stimulated
VAM infection, was higher than that in the control
(Table 3).
Experiment 2.
The intensiy of VAM infection in the rice husk
charcoal plot was 52%, the highest among plots. The intensity in the Bahia grass sod plot was
second highest, whereas that of the abandoned plot was third. The lowest percentage of VAM infec-
tion was in the clean culture plot where herbicides
were used 3 times a year (Table 4). The hyphae, vesicles and arbuscles of VAM fungi were fre-
Table 1. The pH and electric conductivity (EC) of soils treated with charcoal (Experiment 1) .
532 T. Ishii and K. Kadoya
quently observed on/in citrus roots sampled from the charcoal-treated plots (Fig. 1). The SEM photo-
micrographs also indicated that in the charcoal-
treated plot there were many sites where VAM fungi infected and penetrated into the root (Figs. 2
and 3).
Discussion
In Japan, it has long been known that charcoal
is a very effective soil conditioner which promotes
plant growth. Charcoal application may result in
improving physical properties of soil, its fertility,
and biological conditions. The present experiment
indicated that citrus growth and VAM develop-
ment in the root were stimulated by applying char-
coal to soil. This stimulation of citrus growth by charcoal is attributed to an increase in the percen-
tage of VAM infection in the roots. Ogawa (1987)
also reported that the enhanced colonization by
symbiotic microorganisms, such as Rhizobium and VAM fungi, by charcoal application, invigorated
soybean plants.
Table 2. Effect of charcoal application on the growth of satsuma mandarin trees (Experiment 1) .
Table 3. Effect of charcoal application on vesicular-
arbuscular mycorrhizal (VAM) development and
leaf phosphorus (P) concentration in satsuma man-
darin trees (Experiment 1) .
Table 4. Effect of soil management on VAM development in Citrus iyo trees (Experiment 2) .
Fig. 1. Photomicrograph of VAM fungal structures in
Citrus iyo roots stained with typan blue.
a : fungal hyphae ( × 100), b : vesicle ( × 150), c : arbuscle ( × 600).
J. Japan. Soc. Hort. Sci. 63(3) : 529-535. 1994. 533
The increased VAM infections by charcoal ap-
plication may be because charcoal absorbs many kinds of toxic substances and agrochemicals which inhibit root growth and microbial activity. It has
also been shown that some agrochemicals inhibit
the germination of VAM spores (Kobayashi, 1988;
Ogawa, 1987). The growth inhibition of VAM fun-
gi by fungicides such as thiophanate methyl, be-nomyl, iprodione, and copper fungicides is severe. In the case of herbicides, Kobayashi (1988)
showed that the germination of Gigaspora margari-
ta spores was severely repressed by 48 ppm para-
quat dichloride or 410 ppm N - (phosphonomethyl) glycine. In our experiment, the percentage of VAM infcetion in the herbicide-treated clean culture plot is lower than that of the abandoned plot.
The pH value of water extracts from charcoal
was high (Ishii and Kadoya, 1990), indicating that
charcoal ameliorated soil acidity. Generally, soil
pH is low in citrus orchards in Japan, so that the percentage of VAM infection in the root is low and the number of VAM spores in the soil is small
(Ishii et al., 1989b, 1992a). By neutralizing soil acidity, charcoal may be improving the growth and development of VAM fungi.
There are very few reports on VAM develop-
ment in citrus trees grown in Japan. When roots of
satsuma mandarin and Citrus iyo trees from 24 orchards in Ehime prefecture (in southwestern
Japan) were observed for VAM infections, they were not extensive except for an orchard with
good soil conditions which produced 9 ~ 10 t satsu-ma mandarin fruit per 10 a every year (Ishii et al.,
1989b). Numerous VAM spores in the soil and VAM - infected plants are generally observed in
woodlands and non-cultivated fields. This indi-
cates that there are many factors which restrict
Fig. 2. SEM photomicrograph of VAM fungi in Citrus iyo roots.
Left : × 200, Right : × 1000.
Fig. 3. SEM photomicrograph of a VAM spore
(Glomus spp.) and invasion of its hyphae into roots (× 1000).
534 T. Ishii and K. Kadoya
the existence and growth of VAM fungi in
orchards because of our present soil management
practices, such as the usage of agrochemicals and chemical fertilizers. The average annual amount of P applied is about 20 kg per 10 a. That P, espe-
cially soluble P, is detrimental to VAM develop-
ment in citrus roots was reported earlier (Antunes
and Cardoso, 1991; Graham and Timmer, 1985). Several kinds of VAM fungi, however, live in our
soil in spite of many malpractices in our present soil management (Ishii et al., 1992a). We sug-
gested that VAM formation in citrus roots could be effectively increased through application of charcoal to the soil or introduction of a sod cul-
ture system. In particular, the application of char-
coal is very effective for VAM development. Con-trarily, an excess of charcoal inhibits citrus
growth (Ishii and Kadoya, 1990). This inhibition by an excessive application of charcoal might be concerned with an increment of soil pH value.
Therefore, an appropriate amount of charcoal to
be applied is less than 2 t per 10 a (this is approx-imately equivalent to 2% charcoal, Table 2).
Furthermore, such an effect of charcoal may be
strengthened by mixing charcoal and soil. VAM fungi develop well in citrus orchards
where Bahia grass is used for sod (Ishii et al., 1993). We have also indicated that the intensity of
VAM formation on some weeds grown in citrus
orchards was higher than that on citrus trees
(Ishii et al., 1989a). However, sod culture in com-mercial citrus orchards has been unsuccessful in
Japan; most citrus growers believe that a clean culture is best for the production of high-quality fruits. Thus, our soil management system must be
re-evaluated. The prevailing cultural system in which large
quantities of agrochemical and chemical fertilizers are used, should be thoroughly revamped so that a cultural system which maintains beneficial soil
microorganisms is adopted. In conclusion, any ap-
plication of charcoal to the soil is a practical method to improve soil properties and to foster the
development of symbiotic microorganisms includ-
ing VAM fungi.
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炭施用がカンキツの樹体生長およびVA菌 根形成に及ぼす影響
石 井 孝 昭1・ 門 屋 一 臣2
1愛媛 大学教 育学 部790愛 媛 県松 山市 文京 町3
2愛媛 大学 農学部790愛 媛 県 松 山市樽 味3 -5-7
摘 要
炭 施用 が カ ンキ ツの樹体 生 長 お よびVA菌 根 形 成 に
及 ぼす影 響 を調査 した.イ ネ もみが ら,ベ イ ツガ樹 皮
あ るい は カ ンキ ツ ジ ュース かす か ら作 った炭 で処 理 し
た土 壌 を用 いて,ル ー トボ ックス に ウンシ ュ ウ ミカ ン
(カ ラ タチ 台)樹 を 移植 し,こ れ にGlomusfascicula-
tumの胞子 を接種 した.そ の結 果,ボ ックス の ガ ラス
面 に観 察 され る根 の伸長 は,い ず れの炭 施用 区 にお い
て も対照(炭 無 施用)区 よ り旺 盛 であ った.全 生体重,
地 下部 重 お よ び新 梢 重 も炭 施 用 区で増 大 した.VA菌
根 形 成 は対照 区 よ りも炭 施用 区で 良好 であ り,特 に イ
ネ もみ が ら炭 で は その感 染率 が41.5%と 著 しく高 く,
また葉 内の リン含量 も増 加 した.一 方,宮 内 イ ヨカ ン
園 に おけ る炭(イ ネ もみが ら)施 用 区,バ ヒア グ ラス
草生 区,放 任 区お よび慣行裸 地(除 草剤 年3回 使用)
区 のVA菌 根 形成 を比較 調査 した ところ,VA菌 根 菌
の感 染率 は炭 施 用 区(52.0%),バ ヒア グ ラス 草生 区
(16.9%),放 任 区(7.3%),慣 行裸 地 区(3.6%)の
順 で あ った.